Isoflavones represent a class of secondary metabolites produced in legumes by the phenylpropanoid metabolic pathway. The biosynthetic pathway for free isoflavones and their relationship with several other classes of phenylpropanoids is presented in FIG. 1. Many of the enzymes involved in the synthesis of isoflavones in soybean have been identified and the genes in the pathway from phenylalanine ammonia lyase to chalcone synthase and chalcone reductase have been cloned. However, remaining soybean genes involved in synthesis (chalcone isomerase and isoflavone synthase), further metabolism (isoflavone reductase and vestitone reductase), and branch points of the isoflavone pathway that could compete for substrates (flavanone hydroxylase and flavonol synthase) heretofore have not been available.
Free isoflavones rarely accumulate to high levels in soybeans. Instead they are usually conjugated to carbohydrates or organic acids. Soybean seeds contain three types of isoflavones in four different forms: the aglycones daidzein, genistein, and glycitein; the glucosides diadzin, genistin, and glycitin; the acetylgucosides 6"-O-acetyldaidzin, 6"-O-acetylgenistin, and 6"-O-acetylglycitin; and the malonylglucosides 6"-O-malonyldaidzin, 6"-O-malonylgenistin, and 6"-O-malonylglycitin. It has been reported that the isoflavones found in soybean seeds possess antihemolytic (Naim, M. et al. (1976) J.Agric. Food Chem. 24:1174-1177), antifungal (Naim, M. et al. (1974) J Agr. Food Chem. 22:806-810), oestrogenic (Price, K. R. and Fenwick, G. R. (1985) Food Addit. Contam. 2:73-106), tumor suppressing (Messina, M. and Barnes, S. (1991) J. Natl. Cancer Inst. 83:541-546; Peterson, G. et al. (1991) Biochem. Biophys. Res. Commun. 179:661-667), hypolipidemic (Mathur, K. et al. (1964) J. Nutr. 84:201-204), and serum cholesterol lowering (Sharma, R. D. (1979) Lipids 14:535-540) effects. These epidemiological studies indicate that when isoflavone levels are high in soybean seeds and in the subsequent commercial protein products made from the seeds, the dietary intake of these products provide many health benefits.
The content of isoflavones in soybean seeds, however, is quite variable and is affected by both genetics and environmental conditions such as growing location and temperature during seed fill (Tsukamoto, C. et al. (1995) J. Agric. Food Chem. 43:1184-1192; Wang, H. and Murphy, P. A. (1994) J. Agric. Food Chem. 42:1674-1677). In addition, isoflavone content in legumes can be stress-induced by pathogenic attack, wounding, high UV light exposure, and pollution (Dixon, R. A. and Paiva, N. L. (1995) The Plant Cell 7:1085-1097). To date, it has proven difficult to develop soybean lines with consistantly high levels of isoflavones; moreover, lines reported to be low in isoflavone content produced normal levels of isoflavones when grown under standard cultural conditions (Kitamura, K. et al. (1991) Jap. J. Breed. 41:651-654). The isolation and cloning of genes associated with synthesis and metabolism of isoflavones in soybean will afford the application of molecular techniques to achieve stable, high level accumulation of isoflavones.